Two colon carcinoma cell lines (DLD-1 and HCT-15) were established from human tumors; the DLD-1 line was heterogeneous and was cloned into two distinct subpopulations. Cells from these lines and clones were treated in vitro with N,N-dimethylformamide (DMF) and were compared to untreated cells according to two general sets of criteria. One set contains parameters which define a cell as transformed (i.e., anchorage independence and tumorigenicity for nude mice), and the second set contains three antigenic marker systems which would provide evidence that maturation is occurring in treated human colon cancer cells. The colon tissue- or tumor-related markers include carcinoembryonic antigen (CEA), colonic mucoprotein antigen (CMA), and the human blood group determinants. DMF-treated cells are less malignant than are untreated cells; the treated cells show a marked reduction in tumorigenicity and in colongenicity in soft agar. Each of the markers indicates that the treated cells are better differentiated than their untreated counterparts. For example, treated cells show increased expression of normal-CMA and decreased expression of tumor-CMA compared to untreated cells. Cells removed from DMF reverted to express the tumorigenicity, growth properities and antigens characteristic of their untreated counterparts. Therefore, DMF reversibly induces in cultured colon cancer cells a less malignant phenotype with concomitant maturational effects.